To reduce the danger of toxicity
of chloroform, Hara et al.
(Anal Biochem 1978,
90, 420) described
an efficient extraction procedure particularly adapted to nervous tissues.
The tissue sample is homogenized with 18 volumes of a mixture of hexane/2-propanol
(3/2) for 1 minute, the suspension is filtered and the filter rinsed with 3
x 2 vol of the same solvent. As the content of non-lipids is very low (proteins,
pigments, small molecules), the whole liquid phase is evaporated and the dried
extract dissolved (Eder
K et al. Clin Chim
Acta 1993, 219, 93).An adaptation of the Hara's method was demonstrated to be the most efficient
procedure for the extraction of plant sphingolipids (Markham
JE et al., J Biol Chem 2006, 281, 22684).Briefly,
to the frozen tissue, 5 ml of the lower phase of isopropanol/hexane/water, 55/20/25
(v/v) were added. The tissue was disrupted in a glass homogenizer and transferred
to a glass tube which is capped and incubated at 60°C for 15 min with occasional
shaking. After centrifugation, the pellet was extracted twice more, each time
with 5 ml of solvent and the supernatants were combined. A 98% recovery was
obtained with leaf tissue of Arabidopsis, tomato, and soybean.An optimized procedure for extraction
of total lipids from microalgae without disruption and using a chloroform/methanol
mixture has been reported (Ryckebosch E et al., JAOCS 2012, 89, 189).
The recovery of lipids from microalgae by alcohol processing was determined
with two species, Nannochloropsis and Schizochytrium (Wang
G et al., JAOCS 2012, 89, 335).

Extraction
of all lipids with methyl-tert-butyl ether (MTBE).

Accurate profiling of lipidomes were obtained by MTBE extraction which allows
faster and cleaner lipid recovery (Matyash
V et al., J Lipid Res 2010, 49, 1137). Because of MTBE’s low density,
lipid-containing organic phase forms the upper layer during phase separation,
which simplifies its collection and minimizes dripping losses. Nonextractable
matrix forms a dense pellet at the bottom of the extraction tube and is easily
removed by centrifugation. Rigorous testing demonstrated that the MTBE protocol
delivers similar or better recoveries of species of most all major lipid classes
compared with the Folch or Bligh and Dyer recipes.

Briefly, methanol (1.5 ml)
was added to a 200 ml sample aliquot, and the tube was vortexed. Then, 5 ml
of MTBE was added and the mixture was incubated for 1 h at room temperature.
Phase separation was induced by adding 1.25 ml of water. Upon 10 min of incubation
at room temperature, the sample was centrifuged at 1,000 g for 10 min. The upper
organic phase was collected, and the lower phase was re-extracted with 2 ml
of MTBE/methanol/water (10/3/2.5, v/v/v). Combined organic phases were
dried in a vacuum centrifuge. Extracted lipids were dissolved in 200 ml of CHCl3/methanol/water
(60/30/4.5, v/v/v) for storage.

A similar procedure
has been described before analysis of blood lipid classes (Ichihara
K et al., Lipids 2011, 46, 297). This extraction has been also proposed
for the analysis of human brain lipids (Abbott
SK et al., Lipids 2013, 48, 307). This study leads to the conclusion
that this protocol, including a mechanical homogenization utilizing ceramic
beads, is equivalent to the traditional Folch protocol for lipid extraction
and quantification of glycerophospholipid, sphingolipid and sterol species in
human brain tissue.

Extraction of all lipids with hexane

A very precise investigation of the effects of temperature and contact time
on extraction efficiency of sunflower cake was reported using hexane as solvent
(Baümler ER et al., JAOCS 2010, 87, 1489). Extraction at 60°C during
30 min leads to a very high yield (99%) for triacylglycerols and tocopherols
and to a reduced phospholipid extraction (66%).

An alternative to the traditional Folch method
was described using solvent elution of a dry column composed of a tissue sample,
anhydrous sodium sulfate, and Celite diatomaceous earth ground together (Marmer
WN et al. Lipids 1981, 16, 365). Alternatively, lipids may be isolated and
simultaneously separated into neutral and polar fractions by a sequential elution
procedure. Analyses of muscle and adipose tissues demonstrated that results
were similar to those obtained with chloroform/methanol methods.
A less time-consuming dry-column method was scaled down and adapted to 1g samples
(liver and muscle tissues) (Elmer-Frohlich K et al., JAOCS 1992, 69, 243).

Procedure: The sample (about 1 g) is ground for 30 sec. in an ice-chilled
mortar with 4 g anhydrous Na2SO4 and 0.1 ml BHT (20 mg/l
dichloromethane). Celite 545 (3 g) (Fisher Scientific) is added and the mixture
is ground for an additional 30 sec. to obtain a fine homogenized powder.
The powder is poured into a 16 mm X 30 cm glass column packed with glass wool
and 2g of CaHPO4/Celite 545 (1/9 w/w) at its tip. A slight compression
was accomplished at the top with a glass rod. Mortar, pestle, and glass rod
are rinsed with 15 ml of dichloromethane/methanol (9/1, v/v) which are transferred
into the column. In addition to these 15 ml, 50 ml of solvent mixture are added
to elute lipids which are isolated, weighed and analyzed after evaporation of
the solvent under nitrogen flushing.
Multiple columns may be run simultaneously.

A patented process, first reported in 1989 (Barker SA et al., J Chromatogr
1989, 475, 353), known as "matrix solid-phase dispersion" was
described to conduct simultaneously disruption and extraction of solid and semi-solid
samples. Thus, a highly viscous, semi-solid
or solid samplecan be placed in a mortar
containing a bonded-phasesolid support material
(C18 bonded silica) and mechanically blended toperform
a complete disruption and dispersal of thesample.
This blend is sufficientlydry to transfer
and pack into a columnfor more classical
application of solid-phase extraction to the isolationof
sample components. This technique has been most frequently applied to theisolation of drugs, herbicides, pesticides and otherpollutants from animal tissues, fruits and vegetables
(review in : Barker SA, J Chromatogr A 2000, 885, 115).

Bligh and Dyer Method

Bligh and Dyer (Can J Biochem Physiol 1959, 37, 911) introduced a method where extraction and
partitioning are simultaneous, the precipitated proteins are isolated between the two
liquid phases. This method is particularly suitable for lipid extraction of incubation
medium, tissue homogenates or cell suspensions. The extraction can be carried out in a
single tube where previous studies took place.

It must be pointed out
that this extraction method was shown to give significantly lower estimates of
lipid content in samples containing more than 2% lipid (mainly triacylglycerols)
and this underestimation increased with increasing lipid content of the sample (Iverson
SJ et al. Lipids 2001, 36, 1283). Thus, the total lipid content of fatty
samples are accurately determined using the Folch extraction method.

Procedure: To a sample containing 1 ml
water (1 ml cell suspension, homogenized tissue, plasma...), add 3.75 ml of a mixture
chloroform/methanol (1/2) and vortex during 10-15 min, then add 1.25 ml chloroform with
mixing 1 min and 1.25 ml water with mixing another minute before centrifugation. Discard
the upper phase and collect the lower phase through the protein disk with a Pasteur
pipette. For large
volumes of liquid, it is advisable to filter the mixture to remove the insoluble
parts of the sample and to centrifuge the liquid phase to allow the formation of
the two liquid phases. After evaporation, the lipid extract
(lower phase)
will be redissolved in a small volume of
chloroform/methanol (2/1).

The basic procedure was improved to increase the yield of lipids. One of the most common
modifications is to replace water by 1M NaCl. This addition blocked the binding of some
acidic lipids to denatured lipids. If necessary, the addition of 0.2 M phosphoric acid to
the salt solution is possible (Hajra, lipids, 1974, 9, 502) to improve their recovery. In
this case, plasmalogens are converted to lyso lipids.
If an exhaustive extraction is necessary, an
extraction with two steps can be
used.
Similarly, it was described that the addition of acetic acid (0.5% v/v)
in the water phase significantly increased the recovery of acidic phospholipids
(Weerheim AM et al., Anal Biochem 2002, 302, 191). Another modification
has been proposed, in comparison with the traditional extraction method (Jensen
SK, Lipid Technol 2008, 20, 280). Acidification with HCl improved the
extraction of lipids in a shorter time.

Two-step Bligh and Dyer method

To a sample containing 1 ml
water (1.25 g tissue, 1 ml cell suspension, homogenized tissue, plasma...), add 3.75 ml of a mixture
chloroform/methanol (1/2) and vortex during 10-15 min, then add 1.25 ml chloroform with
mixing 1 min and 1.25 ml water with mixing another minute before centrifugation.
Collect the lower phase in another glass tube.
Add 1.88 ml of chloroform to the non-lipid residue, vortex, centrifuge. Mix the
lower phase to the first chloroform phase in the glass tube. After evaporation, the lipid extractwill be dissolved in a small volume of
chloroform/methanol (2/1).

Extraction of
bacteria

An efficient modification of the Bligh and Dyer method, given below, was
proposed for the extraction of lipids from unicellular organisms. Several
parameters were optimized to improve the fatty acid recovery (Lewis T et al.,
J Microbiol Meth 2000, 43, 107). Thus, it was shown that the total amount of
recovered fatty acids increased by about 30% by adding solvents to the
biomass in order of increasing, as opposed to decreasing, polarity.

Cells were harvested by centrifugation at high speed for 15 min. The supernatant
was discarded, the cell pellet re-suspended in 100 ml 1.0% NaCl (w/v), and
re-centrifuged. The second supernatant was discarded and the cell pellet frozen
overnight at -30°C. Frozen biomass was freeze dried for 15 h and subsequently
stored in a sealed glass container at -30°C.
To freeze-dried cells (about 100 mg) to which a total of 114 ml solvents were
added in the sequence: chloroform, methanol, water to achieve a final chloroform/methanol/water
ratio of 1/2/0.8 (v/v/v). Samples were shaken for 15 s immediately following the
addition of each solvent, and allowed to stand for about 18 h, with occasional
shaking by hand.
Phase separation of the biomass-solvent mixtures in the separatory funnels was
achieved by adding chloroform and water to obtain a final chloroform/methanol/water
ratio of 1/1/0.9 (v/v/v). A known portion of each total lipid extract recovered
from the lower chloroform phase was used for further analysis.

Comparing various extraction procedures, it has been shown that a modified
(miniaturized) Bligh and Dyer extraction technique was the most efficient with
an oleaginous bacteria Thraustochytrium sp (Burja AM et al., J Agric
Food Chem 2007, 55, 4795). If only fatty acid determination is required, a
direct saponification using KOH in ethanol was almost as efficient as the
previous one.

At work using a yeast, Yarrowia lipolytica, has reported that bio-based
solvents could be an alternative to petrochemical solvents, such as hexane.
Some differences were noted between experimental and theoretical studies (Breil
C et al., Molecules 2016, 21, 196). Ethyl acetate and methyltetrahydrofuran
are the best candidate solvents to extract all of the lipids of Yarrowia
lipolytica (triglycerides, diglycerides, free fatty acids and
phospholipids) and are derived from renewable resources.

Extraction of highly polar
lipids

When tissues are rich in highly polar lipids
such as gangliosides, a reliable extraction method is needed to prevent their loss while
extracting all other lipid classes. All the previously described techniques brings
gangliosides and likely a part of other very polar lipids into the water-rich layer. A
method, recently described for nervous tissues (Dreyfus et al., Anal Biochem 1977, 249, 67-78), prevents these drawbacks.
Small tissue samples corresponding up to 10 mg protein suspended in 0.5 ml water are mixed
with 5 ml chloroform/methanol mixture (1/1) for 30 min. The pellet obtained by
centrifugation is extracted again with successively 3 ml of the same solvent, 3 ml of a
mixture chloroform/methanol (1/2) and 3 ml of a mixture chloroform/methanol/water
(60/30/4.5). The four lipid extracts are combined and evaporated and the dry residue
dissolved for further purification.

A liquid/liquid extraction has been
used with success for mass spectroscopy estimation of brain gangliosides (Garcia
AD et al., J Chromatogr B 2014, 947-8, 1). The lipid extract was suspended
in a mixture of chloroform/methanol/water (30/60/8, v/v/v), vortexed and sonicated.
The solution was then centrifuged and the aqueous phase containing the enriched
gangliosides was collected and set aside while the organic phase was then subjected
to the same extraction procedure. The supernatants were combined and dried under
vacuum. Once dry, the lipid extracts were resuspended in 10 mL
of the liquid chromatography starting buffer. The gangliosides were purified
on C8 SPE cartridges before LC-MS analysis.

A very efficient method has been developed to isolate and purify GM1 from pig
brain (Bian
L et al., Biomed Chromatogr 2015, 29, 1604). The method consisted of
a precipitation by acetone followed by an extraction by chloroform–methanol–water.
The purification was done using a two-step chromatographic separation by DEAE–Sepharose
Fast Flow anion-exchange medium and Sephacryl S-100 HR size-exclusion medium.
The final yield of GM1 was about 0.022% ( g/g) with the purity of about 98%.
This method will probably provide a reference alternative for isolation and
purification of other amphipathic substances in biological tissues. Lyso derivatives (N-deacylated) of glycosphingolipids
are not efficiently recovered from cell extracts because of their high polarity.
The glycosylsphingosine are found in tissues from patients and in animal
models. In normal conditions, their concentration are very low and needs very
efficient techniques to be evaluated. It was shown that a second extraction
with water-saturated butanol of the upper aqueous phase obtained after a Folch
extraction was necessary to recover up to 98% of the tissue lysoglycosphingolipids
(Bodennec
J et al., J Lipid Res 2003, 44, 218).

Extraction of
plasma total lipids

For the extraction of plasma lipids
we used a very rapid and efficient method.
To 0.2 ml plasma add 0.3 ml 0.5 M KH2PO4 , 1.5 ml chloroform
and 0.5 ml methanol. After vortexing 2 minutes and centrifugation, the lower
phase is collected with a Pasteur pipette through the protein disk and evaporated.

A procedure using the detergent Triton
X-114 was shown to be very efficient for the extraction of plasma lipids, while
sparing the protein fraction for further use (Ferraz TPL et al., J Biochem
Biophys Meth 2004, 58, 187).

A method based on the modification of an extraction method originally developed
for pesticide residue analysis in food has been described for the purpose of
isolating lipids from biological fluids (plasma, urine) (Bang
DY et al., J Chromatogr A 2014, 1331, 19). The procedure adapted for
the preparation of samples for mass speectrometry includes a extraction/partitioning
step with a mixture of CHCl3/CH3OH in the presence of
MgSO4 and CH3COONa and an adsorption/desorption step with
C18 particles. That method was applied to lipid extracts from both human urine
and plasma samples, demonstrating that it can be powerfullyutilized for high-speed
(<15 min) preparation of lipids compared to the Folch method, with equivalent
or slightly improved results in lipid identification using liquid chromatography
and mass spectrometry.Delipidation of plasma,
serum or plant seeds

When plasma proteins, including the
apolipoproteins, must be preserved from denaturation during the extraction of
lipids a specific solvent system must be used (Cham BE et al., J Lipid Res
1976, 17, 176).
The most common procedure used for delipidation of plasma, protein solutions
or cell culture medium involves the extraction of all kinds of lipids with a
mixture of butanol and di-isopropyl ether. The proteins remain in solution in
the aqueous phase, while the organic phase contains the dissolved lipids.

Procedure : One volume of serum or plasma containing 0.1 mg/ml of ethylenediamine
tetraacetate (EDTA) is added to 2 volumes of a mixture of butanol/di-isopropyl
ether (40/60, v/v). The vials are tightly closed and fastened on a mechanical
rotator providing end-over-end rotation at about 30 rpm for 0.5 h.
After extraction, the mixture is centrifuged at low speed (2000 rpm) for 2 min
to separate the aqueous and organic phase. The aqueous phase containing the
delipidated proteins is removed by careful suction with needle and pump or syringe.
Traces of butanol remaining in the aqueous solution may be removed if necessary
by washing that phase with 2 volumes of di-isopropyl ether. Residual solvent
may be removed by an extraction with a water pump aspirator at 37°C for some
minutes.

When proteins from plant oilseeds are studied by electrophoresis, lipids must
be removed to prevent important interferences and thus to obtain good resolution.
It was demonstrated that, in the presence of chloroform methanol, lipid contaminants
can be thoroughly removed by the combination of two precipitation steps (10%
TCA/acetone and acetone) and aqueous TCA wash steps (Wang W et al., Anal
Biochem 2004, 329, 139).

Several procedures were described
to extract the polyphosphoinositides since they are known to bind strongly to
proteins during their denaturation. To improve the recovery, the use of an acidic
solution is necessary. We used theprocedure ofHoneyman (Biochem J 1983,
212, 489) which is a modification of the Lloyd's method
(Br J Haematol 1972, 23, 571-585).
This is the one-step method of Bligh and Dyer modified by the inclusion of HCl
to improve recovery of acidic phospholipids :

1 ml of cell suspension is mixed with 3.75 ml of chloroform/methanol/12N HCl
(2/4/0.1, v/v). After thorough mixing, 1.25 ml of chloroform is added with vortexing
30 sec followed by 1.25 ml of water with similar mixing. After centrifugation
10 min at low speed, the lower chloroform layer is removed and transferred to
a glass tube for evaporation.

Method used to extract platelet
phosphoinositides

Stop incubation of the cell suspension by adding 1 ml of chloroform/methanol
(1/1) to 1.5 ml of aqueous medium and vortex some seconds. Transfer the mixture
with a Pasteur pipette into a 15 ml Falcon polypropylene tube.
Add to the mixture 4 ml of chloroform/methanol (1/1), then 0.4 ml 10N HCl and
then 0.5 ml water. Vortex during 5 min to extract lipids and centrifuge the
plastic tube at low speed 10 min at 4°C.
Transfer the lower phase into a second Falcon tube by sampling through the proteinaceous
disk with a Pasteur pipette. Add to this solution 2.5 ml methanol, 2.1 ml water
and 0.4 ml 10N HCl. Vortex some seconds and centrifuge at low speed in the cold.
Transfer the lower phase as previously in a glass tube, evaporate with the help
of nitrogen and dissolve the residue with a small volume of chloroform/methanol
(1/1).

Extraction of
plant material

Plant tissues are difficult to extract because of active lipases which hydrolyze
rapidly phospholipids glycolipids and increase the amount of free fatty acids
in the extract. Thus, a solvent frequently used to inhibit these enzymes is
isopropanol.
Nichols' method: Plant tissues are minced and macerated with 100 parts
(w/w) of isopropanol. The mixture is filtered, the solid is extracted again
with 200 parts of chloroform/isopropanol mixture (1/1, v/v). The combined filtrates
are evaporated, dissolved in a small volume of chloroform/methanol (2/1, v/v)
and, if necessary, washed according the Folch's procedure.
Optimal conditions of extraction of microalgae with aqueous isopropanol combined
or not with cell rupture has been reported (Yao L et al., JAOCS 2013, 90,
571). The oil yield was largely increased after ultrasonic cell rupture
after extraction with 88 or 95% isopropanol.

The extraction of algae is made with hot isopropanol (60°C) added to
the cell suspension. It appears that unicellular algae (plankton) must be extracted
rapidly with a minimum of preparative mechanical treatments (centrifugation,
filtration). A preliminary small scale extraction is recommended to choose the
procedure to be adopted.
A comparative evaluation of several extraction methods was done in three types
of macroalgae (Kumari
P et al., Anal Biochem 2011, 415, 134). Care should be taken while selecting
the method for macroalgae, according to the group to which they belong, otherwise
there would be a risk of obtaining erratic and inaccurate results.
The extraction of neutral lipids from microalgae has been efficiently done on
lyophilized material (Fajardo AR et al., Eur J Lipid Sci Technol 2007, 109,
120). Briefly, first, 96% ethanol was used to extract the lipids from the
dry biomass. Second, a biphasic system was formed by adding water and hexane
to the extracted crude oil. Thus, the purified lipids were transferred to the
hexane phase while most impurities remained in the aqueous phase.
An overview of advances made in technologies for extracting microalgae oil may
be consulted before doing experiments (Mercer P et al., Eur J Lipid Sci Technol
2011, 113, 539). Solvent extraction technologies with extraction alternatives
such as mechanical milling and pressing, enzymatic and supercritical fluid extraction
are compared.
A comparative study of various extraction treatments have been reported using
the microalgae Spirulina (Zheng G et al., JAOCS 2012, 89, 561). A purification
was used to remove the pigments from the extracted lipids.

Because of their large hydrophilic
polar head, solubilisation of glycosylated sphingolipids in usual organic solvents
is inaccurate. When doing a phase partition in chloroform/methanol/water mixtures,
these lipids remain insoluble for the most part or are recovered in the aqueous
phase and interphase. A new extraction method to purify total plant sphingolipids
has been developed (Buré C et al., Rapid Commun. Mass Spectrom 2013, 25,
3131). Briefly, plants cells were blended with cold 0.1 N aqueous acetic
acid in a chilled Waring Blendor. The slurry was filtered under vacuum and the
aqueous acetic acid filtrate was discarded. The residue was then re-extracted
with hot 70% ethanol containing 0.1 N HCl. The filtrate was chilled and left
at room temperature overnight. The precipitate was pelleted by centrifugation.
The sphingolipid-enriched pellet was washed twice with cold acetone, and twice
with cold diethyl ether to yield a whitish precipitate. Glycosylated sphingolipid
contained in the precipitate were then dissolved in tetrahydrofuran (THF)/methanol/water
(4/4/1, v/v/v) containing 0.1% formic acid by heating at 60°C, followed by gentle
sonication. This solution was further used for mass spectrometry analyses (Buré
C et al., Rapid Commun Mass Spectrom 2011, 25, 3131; Cacas
JL et al., Phytochemistry 2013, 96, 191).

Enzyme-assisted aqueous extraction (EAEP)

EAEP has been employed to extract
different compounds from plants, and has been proved to be effective in improving
the yield of various components. Improved lipid extraction was observed in many
different oil-bearing plant materials including soybean (Freitas SP et al.,
Fett-Lipid 1997, 99, 333), sunflower
seeds (Sineiro J et al., Food Chem 1998, 61, 467) and sesame (Latif
S et al., Food Chem. 2011, 125, 679).14 In addition, EAEP will make it possible
to extract and separate oil directly from algae in the natural aqueous environment
of algae cultures, which avoids the collection and drying process of algae biomass.
As insoluble nonhydrolyzable biopolymers (algaenans) are present in cell wall
of algae, the EAEP methods established for common terrestrial plants cannot
be applied directly to the lipid extraction from microalgae. Improvement in
lipid extraction of these vegetals has been done in using sonication combined
with enzyme treatment (Liang K et al., J Agric Food Chem 2012, 60, 11771).

Oilseeds may be analyzed for oil content by an exhaustive extraction
with petroleum ether. A comparison of five methods to measure the oil contents
in oilseeds may be studied before choosing a specific procedure (Barthet
VJ et al., J Oleo Sci 2002, 51, 589).

A one-tep extraction has been described to study the composition
of triglycerides in small piece of seed and is suitable for a large number
of tissue samples should be examined as in selecting new plant varieties. This
easy and reliable method is based on an incubation of samples (20-50 mg) without
shaking in a mixture containing heptane / 0.17 M NaCl in methanol (66.6/33.3,
v/v), for 2 h at 80°C. After cooling, the upper phase containing mainly triglycerides
was transferred to a new test tube for further analysis. Even under incomplete
triglyceride extraction (80% maximum) the triglyceride ecomposition is representative
of the total triglycerides found in the tissue (Ruiz-Lopez N et al., Anal
Biochem 2003, 317, 247).

An efficient surfactant-based extraction of corn oil from corn germ has been
proposed (Kadioglu SI et al., JAOCS 2011, 88, 863). Hexane and/or other
organic solvents were avoided in the process. Greater than 80% of corn oil can
be extracted with low surfactant and salt concentrations. It was concluded that
aqueous-based surfactant microemulsion oilseed extraction is a promising alternative
approach for oil extraction.

The extraction of xanthophyll was shown to be improved using cellulolytic enzymes
and highly competitive when compared to the traditional process of pigment extraction
(Navarrete-Bolanos JL et al., J Agric Food Chem 2004, 52, 3394). These
data may foster the development of new extraction procedures in plants based
on previous enzymatic hydrolysis of cell membranes.

The extraction of flour can be made with hexane at room temperature but the
efficiency of phospholipid recovery is dependent upon the temperature and the
moisture content (Snyder HE, Inform 2004, 15, 575).

The tiny piece of tissue is directly applied in a small hole or groove made
in the concentration zone (kieselgur preadsorbent) of the TLC plate (cf Whatman
plates). This plate is then rapidly applied onto a steel plate pre-cooled in
liquid nitrogen. After one or two minute freezing, the plate is lyophilized
one or two hours under vacuum. The plate is then rapidly submitted to a solvent
elution adapted to the desired analysis.

Mineralized
samples

As some acidic phospholipids (mainly phosphatidylserine) are known to complex
with calcium, particularly in the presence of Pi, a complete extraction of membrane
lipids in mineralized tissues must be done after chemical demineralization (Wu
L et al., J Biol Chem 2002, 277, 5126). This approach was first discovered by
Shapiro IM in 1971 (Arch oral Biol 1971, 16, 411; Calcif Tissue
Res 1970, 5, 21). A complete extraction and analysis of mineralized bone
tissue and bone marrow lipids have been described in detail (During
A, J Chromatogr A 2017, 1515, 232).

Procedure:

Small tissue samples are powdered in the cold (Freezer mill)
and extracted with chloroform/methanol (2/1) mixture. After a short sonication
(1-2 min), the suspension is centrifuged (3000 rpm, 12 min) and the extract
collected. The pellet is demineralized with 0.5M Na salt EDTA for 20-30 min
and sedimented after a short centrifugation. After removal of the supernatant,
the decalcified residue (pellet) is reextracted using a chloroform/methanol/conc.
HCl (200/100/1) mixture. The two lipid extracts are mixed, dried and washed
with saline to remove non-lipid contaminants.

Skin
surface

Several lipids are present at the
surface of mammalian skin. Their analysis is of great interest in relation with
medical treatment in diseases such as acne, atopic dermatitis, seborrhea or
psoriasis. Applications may be extended to cosmetic and alimentary fields. The
extraction of surface lipids is efficiently processed with a very simple design
(Michael-Jubeli
R et al., J Lipid Res 2011, 52, 143).
Briefly,two lipid-free absorbent papers are
maintained on the defined area for 30 minutes using a medical tape, and then
removed with tweezers and introduced into a closed vial. This step was repeated
four times. The collected lipids are extracted from papers twice with 40 ml of
diethyl ether. The solution is concentrated and transferred into a vial.

A novel method for the extraction of insect cuticular hydrocarbons has been
described (Choe DH et al., J Chem Ecol 2012, 38, 176). The cuticular
hydrocarbons are first adsorbed to solica gel and then are eluted using organic
solvents.